U.S. patent number 6,908,615 [Application Number 08/913,644] was granted by the patent office on 2005-06-21 for dna encoding human papilloma virus type 18.
This patent grant is currently assigned to Merck & Co., Inc.. Invention is credited to Hugh A. George, Kathryn J. Hofmann, Kathrin U. Jansen, Joseph G. Joyce, Michael P. Neeper.
United States Patent |
6,908,615 |
Hofmann , et al. |
June 21, 2005 |
**Please see images for:
( Certificate of Correction ) ** |
DNA encoding human papilloma virus type 18
Abstract
The present invention is directed to DNA molecules encoding
purified human papillomavirus type 18 and derivatives thereof.
Inventors: |
Hofmann; Kathryn J.
(Collegeville, PA), Jansen; Kathrin U. (Fort Washington,
PA), Neeper; Michael P. (Collegeville, PA), Joyce; Joseph
G. (Lansdale, PA), George; Hugh A. (Schwenksville,
PA) |
Assignee: |
Merck & Co., Inc. (Rahway,
NJ)
|
Family
ID: |
34654479 |
Appl.
No.: |
08/913,644 |
Filed: |
November 21, 1997 |
PCT
Filed: |
March 18, 1996 |
PCT No.: |
PCT/US96/03649 |
371(c)(1),(2),(4) Date: |
November 21, 1997 |
PCT
Pub. No.: |
WO96/29413 |
PCT
Pub. Date: |
September 26, 1996 |
Current U.S.
Class: |
424/204.1;
424/186.1; 435/235.1; 435/320.1; 435/6.14; 435/69.1; 530/300 |
Current CPC
Class: |
C07K
14/005 (20130101); A61K 2039/5258 (20130101); C12N
2710/20022 (20130101) |
Current International
Class: |
A61K
39/12 (20060101); A61K 039/12 () |
Field of
Search: |
;424/204.1,186.1
;435/320.1,6,69.1,235.1 ;530/300 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 456 197 |
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May 1991 |
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EP |
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0 256 321 |
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Jun 1989 |
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WO |
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WO 93/02184 |
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Feb 1993 |
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WO |
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WO 94/00152 |
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Jan 1994 |
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WO |
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WO 94/05792 |
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Mar 1994 |
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WO |
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WO 94/20137 |
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Sep 1994 |
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WO |
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Other References
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Papillomavirus Type 16 by Expression in a Vaccinia Virus
Recombinant", J. Gen. Virol., vol. 69, pp. 1263-1273 (1988). .
Doorbar, et al., "Identification of Proteins Encoded by the L1 and
L2 Open Reading Frames of Human Papillomavirus 1a", J. of Virol.,
vol. 61, No. 9, pp. 2793-2799 (Sep., 1987). .
Hagensee, et al., "Self-Assembly of Human Papillomavirus Type 1
Capdids by Expression o the L1 Protein Alone or by Coexpression of
the L1 and L2 Capsid Proteins", J. of Virology, pp. 315-322 (Jan.
1993). .
Kirnbauer, et al., "Papillomavirus L1 major capsid protein
self-assembles into virus-like particles that are highly
immunogenic", Proc. Natl. Acad. Sci., vol. 89, pp. 12180-12184
(Dec. 1992). .
Cann, et al., "Self-assembly of human papillomavirus type 16
capsids by expression of the L1 protein in insect cells", FEMS
Microbiology Letters, vol. 117, pp. 267-274 (1994). .
Lin, et al., "Effective Vaccination against Papillomavirus
Development by Immunization with L1 or L2 Structural Protein of
Cottontail Rabbit Papillomavirus", Virology, vol. 187, pp. 612-619
(1992). .
Rose, et al., "Expression of Human Papillomavirus Type 11 L1
Protein in Insect Cells: In Vivo and In Vitro Assembly of Viruslike
Particles", J. of Virol., pp. 1936-1944 (Apr. 1993). .
Steele, et el., Humoral Assays of Human Sera to Disrupted and
Nondisrupted Epitopes and Human Papillomavirus Type 1., Virology,
vol. 174, pp. 388-398 (1990). .
Strike, et al., "Expression of Escherichia col of Seven DNA
Fragments Comprising the Complete L1 and L2 Open Reading Frames of
Human Papillomavirus Type 6b . . . ", J. Gen. Virol., vol. 70, pp.
543-555 (1989). .
Zhou, et al., "Synthesis and assembly of infectious bovine
papillomavirus particles in vitro", J. of Gen. Virol., vol. 74, pp.
763-768 (1993). .
Zhou, et al., "Expression of Vaccinia Recombinant HPV 16 L1 and L2
ORF Proteins in Epithelial Cells Is Sufficient for Assembly of HPV
Virion-Like Particles", Virology, vol. 185, pp. 251-257 (1991).
.
Zhou, et al., "Increased antibody responses to human papillomavirus
type 16 L1 protein expressed by recombinant vaccinia lacking serine
protease inhibitor genes", J. of Gen. Virol., vol. 71, pp.
2185-2190 (1990). .
Sasagawa, et al., "Synthesis and Assembly of Virus-Like Particles
of Human Papillomaviruses Type 6 and Type 16 in Fission Yeast
Schizosaccharomyces pombe", Virology., vol. 206, pp. 126-136
(1995). .
Cole, et al., "Nucleotide Sequence and Comparative Analysis of the
Human Papillomavirus Type 18 Genome", J. Mol. Biol., vol. 93, pp.
599-608 (1987). .
Kaufman, "Vectors Used for Expression in Mammalian Cells", Methods
in Enzymology, vol. 185, pp. 487-511 (1990). .
Xi, et al., "Baculovirus expression of the human papillomarirus
type 16 capsid proteins: detection of L1-L2 protein complexes",
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|
Primary Examiner: Salimi; Ali R.
Attorney, Agent or Firm: Finnegan; Alysia A. Giesser; Joanne
M.
Parent Case Text
CROSS-RELATED TO OTHER APPLICATIONS
This application is a 371 of PCT/US96/03649, international filing
date of Mar. 18, 1996, which claims priority to U.S. Ser. No.
08/408,669, filed Mar. 22, 1995, now issued as U.S. Pat. No.
5,840,306, and to U.S. Ser. No. 08/409,122, filed Mar. 22, 1995,
now issued as U.S. Pat. No. 5,820,870.
Claims
What is claimed is:
1. An essentially purified HPV18 L1 protein comprising a sequence
of amino acids as set forth in SEQ ID NO:2.
2. Virus-like particles comprised of recombinant L1 protein, or
recombinant L1+L2 proteins of human papillomavirus 18, wherein the
L1 protein comprises a sequence of amino acids as set forth in SEQ
ID NO:2 and the L2 protein comprises a sequence of amino acids as
set forth in SEQ ID NO:4, wherein the recombinant L1 protein or the
recombinant L1+L2 proteins are produced in yeast.
3. A method of producing the virus-like particles of claim 2,
comprising: (a) transforming yeast with a recombinant DNA molecule
encoding papillomavirus L1 protein or papillomavirus L1+L2
proteins; wherein the L1 protein comprises a sequence of amino
acids as set forth in SEQ ID NO:2 and the L2 protein comprises a
sequence of amino acids as set forth in SEQ ID NO:4 (b) cultivating
the transformed yeast under conditions that permit expression of
the recombinant DNA molecule to produce the recombinant
papillomavirus protein; and (c) isolating the recombinant
papillomavirus protein to produce the virus-like particles of claim
2.
4. Recombinant papillomavirus protein produced by the method of
claim 3.
5. A vaccine comprising the virus-like particles of claim 2 and a
pharmaceutically acceptable carrier.
6. Pharmaceutical compositions comprising the virus-like particles
of claim 2 and a pharmaceutically acceptable carrier.
7. A method of preventing papillomavirus infection comprising
administering the vaccine of claim 5 to a host.
8. A method for inducing an immune response in an animal comprising
administering the virus-like particles of claim 2 to an animal.
Description
FIELD OF THE INVENTION
The present invention is directed to DNA molecules encoding
purified human papillomavirus type 18 and derivatives thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the HPV18 L1 nucleotide (SEQ ID NO:1) and deduced
amino acid (SEQ ID NO:2) sequences.
FIG. 2 is a list of amino acid variations within the L1 protein of
HPV 18.
FIG. 3 shows the HPV18 L2 nucleotide (SEQ ID NO:3) and deduced
amino acid (SEQ ID NO:4) sequences.
FIG. 4 shows an immunoblot of HPV18 L1 protein expressed in
yeast.
FIG. 5 shows an immunoblot of HPV18 L2 protein expressed in
yeast.
FIG. 6 is an election micrograph of virus-like particles formed by
HPV18 L1 protein expressed in yeast.
BACKGROUND OF THE INVENTION
Papillomavirus (PV) infections occur in a variety of animals,
including humans, sheep, dogs, cats, rabbits, monkeys, snakes and
cows. Papillomaviruses infect epithelial cells, generally inducing
benign epithelial or fibroepithelial tumors at the site of
infection. PV are species specific inflective agents; a human
papillomavirus does not infect a nonhuman animal.
Papillomaviruses may be classified into distinct groups based on
the host that they infect. Human papillomaviruses (HPV) are further
classified into more than 70 types based on DNA sequence homology.
PV types appear to be type-specific immunogens in that a
neutralizing immunity to infection by one type of papillomavirus
does not confer immunity against another type of
papillomavirus.
In humans, different HPV types cause distinct diseases. HPV types
1, 2, 3, 4, 7, 10 and 26-29 cause benign warts in both norrnal and
immunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17,
19-25, 36 and 46-50 cause flat lesions in immunocompromised
individuals. HPV types 6, 11, 34, 39, 41-44 and 51-55 cause benign
condylomata of the genital or respiratory mucosa. HPV types 16 and
18 cause epithelial dysplasia of the genital mucosa and are
associated with the majority of in situ and invasive carcinomas of
the cervix, vagina, vulva and anal canal.
Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral
DNA viruses that encode for up to eight early and two late genes.
The open reading frames (ORFs) of the vines genomes are designated
E1 to E7 and L1 and L2, where "E" denotes early and "L" denotes
late. L1 and L2 code for virus capsid proteins. The early (E) genes
are associated with functions such as viral replication and
cellular transformation.
The L1 protein is the major capsid protein and has a molecular
weight of 55-60 kDa. The L2 protein is a minor capsid protein which
has a predicted molecular weight of 55-60 kDa and an apparent
molecular weight of 75-100 kDa as determined by polyacrylamide gel
electrophoresis. Immunological data suggest that most of the L2
protein is internal to the L1 protein within the viral capsomere.
The L1 ORF is highly conserved among different papillomaviruses.
The L2 proteins are less conserved among different
papillomaviruses.
The L1 and L2 genes have been identified as good targets for
immunoprophylaxis. Studies in the cottontail rabbit papillomavirus
(CRPV) and bovine papillomavirus (BPV) systems have shown that
immunizations with the L1 and L2 proteins expressed in bacteria or
by using vaccinia vectors protected animals from viral infection.
Expression of papillomavirus L1 genes in baculovirus expression
systems or using vaccinia vectors resulted in the assembly of
virus-like particles (VLP) which have been used to induce
high-titered virus-neutralizing antibody responses that correlate
with protection from viral challenge.
Following HPV type 16, HPV18 is the second most prevalent HPV type
found in cervical carcinomas. HPV18 was detected in 5-20% of
cervical cancer biopsies collected from various parts of the world
(Ikenberg, H. 1990. Human papillomavirus DNA in invasive genital
carcinomas. In Genital Papillomavirus Infections, C. Gross et aL,
eds. p. 85-112). There appears to be a geographic dependence of
infection with HPV18 since tumor biopsies from African and South
American women harbor HPV18 more frequently than similar biopsies
from European and North American women. The underlying reasons for
these geographic differences are not known. The development of a
vaccine against HPV18 infection becomes extremely relevant since
HPV18 is also associated with more aggressively growing cancers and
is rarely found in the milder precursor lesions, CIN I-II.
SUMMARY OF THE INVENTION
The present invention is directed to DNA molecules encoding
purified human papillomavirus type 18 (HPV type 18; HPV18) and uses
of the DNA molecules.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to DNA molecules encoding
purified human papillomavirus type 18 (HPV type 18; HPV18) and
derivatives thereof. Such derivatives include but are not limited
to peptides and proteins encoded by the DNA, antibodies to the DNA
or antibodies to the proteins encoded by the DNA, vaccines
comprising the DNA or vaccines comprising proteins encoded by the
DNA, immunological compositions comprising the DNA or the proteins
encoded by the DNA, kits containing the DNA or RNA derived from the
DNA or proteins encoded by the DNA.
Pharmaceutically useful compositions comprising the DNA or proteins
encoded by the DNA may be formulated according to known methods
such as by the admixture of a pharmaceutically acceptable carrier.
Examples of such carriers and methods of formulation may be found
in Remington's Pharmaceutical Sciences. To form a pharmaceutically
acceptable composition suitable for effective administration, such
compositions will contain an effective amount of the DNA or protein
or VLP. Such compositions may contain DNA or proteins or VLP
derived from more than one type of HIPV.
Therapeutic or diagnostic compositions of the invention are
administered to an individual in amounts sufficient to treat or
diagnose PV infections. The effective amount may vary according to
a variety of factors such as the individual's condition, weight,
sex and age. Other factors include the mode of administration.
Generally, the compositions will be administered in dosages ranging
from about 1 .mu.g to about 1 mg.
The pharmaceutical compositions may be provided to the individual
by a variety of routes such as subcutaneous, topical, oral,
mucosal, intravenous and intramuscular.
The vaccines of the invention comprise DNA, RNA or proteins encoded
by the DNA that contain the antigenic determinants necessary to
induce the formation of neutralizing antibodies in the host. Such
vaccines are also safe enough to be administered without danger of
clinical infection; do not have toxic side effects; can be
administered by an effective route; are stable; and are compatible
with vaccine carriers.
The vaccines may be administered by a variety of routes, such as
orally, parenterally, subcutaneously, mucosally, intravenously or
intramuscularly. The dosage administered may vary with the
condition, sex, weight, and age of the individual; the route of
administration; and the type PV of the vaccine. The vaccine may be
used in dosage forms such as capsules, suspensions, elixirs, or
liquid solutions. The vaccine may be formulated with aN
immunologically acceptable carrier.
The vaccines are administered in therapeutically effective amounts,
that is, in amounts sufficient to generate a immunologically
protective response. The therapeutically effective amount may vary
according to the type of PV. The vaccine may be administered in
single or multiple doses.
The DNA and DNA derivatives of the present invention may be used in
the formulation of immunogenic compositions. Such compositions,
when introduced into a suitable host, are capable of inducing an
immune response in the host.
The DNA or its derivatives may be used to generate antibodies. The
term "antibody" as used herein includes both polyclonal and
monoclonal antibodies, as well as fragments thereof, such as, Fv,
Fab and F(ab)2 fragments that are capable of binding antigen or
hapten.
The DNA and DNA derivatives of the present invention may be used to
serotype HPV infection and HPV screening. The DNA, recombinant
proteins, VLP and antibodies lend themselves to the formulation of
kits suitable for the detection and serotyping of HPV. Such a kit
would comprise a compartmentalized carrier suitable to hold in
close confinement at least one container. The carrier would further
comprise reagents such as HPV18 DNA, recombinant HPV protein or VLP
or anti-HPV antibodies suitable for detecting a variety of HPV
types. The carrier may also contain means for detection such as
labeled antigen or enzyme substrates or the like.
The DNA and derived proteins therefrom are also useful as molecular
weight and molecular size markers.
Because the genetic code is degenerate, more than one codon may be
used to encode a particular amino acid, and therefore, the amino
acid sequence can be encoded by any of a set of similar DNA
oligonucleotides. Only one member of the set will be identical to
the HPV18 sequence but will be capable of hybridizing to HPV18 DNA
even in the presence of DNA oligonucleotides with mismatches under
appropriate conditions. Under alternate conditions, the mismatched
DNA oligonucleotides may still hybridize to the HPV18 DNA to permit
identification and isolation of HPV18 encoding DNA.
The purified HPV18 DNA of the invention or fragments thereof may be
used to isolate and purify homologues and fragments of HPV18 from
other sources. To accomplish this, the first HPV18 DNA may be mixed
with a sample containing DNA encoding homologues of HPV18 under
appropriate hybridization conditions. The hybridized DNA complex
may be isolated and the DNA encoding the homologous DNA may be
purified therefrom.
It is known that there is a substantial amount of redundancy in the
various codons which code for specific amino acids. Therefore, this
invention is also directed to those DNA sequences which contain
alternative codons which code for the eventual translation of the
identical amino acid. For purposes of this specification, a
sequence bearing one or more replaced codons will be defined as a
degenerate variation. Also included within the scope of this
invention are mutations either in the DNA sequence or the
translated protein which do not substantially alter the ultimate
physical properties of the expressed protein. For example,
substitution of valine for leucine, arginine for lysine, or
asparagine for glutamine may not cause a change in functionality of
the polypeptide.
It is known that DNA sequences coding for a peptide may be altered
so as to code for a peptide having properties that are different
than those of the naturally-occurring peptide. Methods of altering
the DNA sequences include, but are not limited to site-directed
mutagenesis.
As used herein, a "functional derivative" of HPV18 is a compound
that possesses a biological activity (either functional or
structural) that is substantially similar to the biological
activity of HPV18. The term "functional derivatives" is intended to
include the "fragments," "variants," "degenerate variants,"
"analogs" and "homologues" or to "chemical derivatives" of HPV18.
The term "fragment" is meant to refer to any polypeptide subset
ofHPV18. The term "variant" is meant to refer to a molecule
substantially similar in structure and function to either the
entire HPV18 molecule or to a fragment thereof. A molecule is
"substantially similar" to HPV18 if both molecules have
substantially similar structures or if both molecules possess
similar biological activity. Therefore, if the two molecules
possess substantially similar activity, they are, considered to be
variants even if the structure of one of the molecules is not found
in the other or even if the two amino acid sequences are not
identical.
The term "analog" refers to a molecule substantially similar in
function to either the entire HPV18 molecule or to a fragment
thereof.
A variety of procedures may be used to molecularly clone HPV18 DNA.
These methods include, but are not limited to, direct functional
expression of the HPV1 8 genes following the construction of a
HPV18-containing CDNA or genomic DNA library in an appropriate
expression vector system. Another method is to screen HPV18
containing cDNA or genomic DNA library constructed in a
bacteriophage or plasmid shuttle vector with a labeled
oligonucleotide probe designed from the amino acid sequence of the
HPV18. An additional method consists of screening a HPV1
8-containing cDNA or genomic DNA library constructed in a
bacteriophage or plasmid shuttle vector with a partial DNA encoding
the HPV18. This partial DNA is obtained by the specific polymerase
chain reaction (PCR) amplification of HPV18 DNA fragments through
the design of degenerate oligonucleotide primers from the amino
acid sequence of purified HPV18. Another method is to isolate RNA
from HPV18-producing cells and translate the RNA into protein via
an in vitro or an in vivo translation system. The translation of
the RNA into a peptide or a protein will result in the production
of at least a portion of HPV18 protein which can be identified by,
for example, the activity of HPV18 protein or by immunological
reactivity with an anti-HPV18 antibody. In this method, pools of
RNA isolated from HPV18-producing cells can be analyzed for the
presence of an RNA which encodes at least a portion of the HPV18.
Further fractionation of the RNA pool can be done to purify the
HPV18 RNA from non-HPV18 RNA. The peptide or protein produced by
this method may be analyzed to provide amino acid sequences which
in turn are used to provide primers for production of HPV18 cDNA,
or the RNA used for translation can be analyzed to provide
nucleotide sequences encoding HPV18 and produce probes for the
screening of a HPV18 cDNA library. These methods are known in the
art and can be found in, for example, Sambrook, J., Fritsch, E. F.,
Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. 1989.
It is apparent that other types of libraries, as well as libraries
constructed from other cells or cell types, may be useful for
isolating HPV18-encoding DNA. Other types of libraries include, but
are not limited to, cDNA libraries derived from other cells or cell
lines containing HIPV type 18 and genomic DNA libraries.
Preparation of cDNA libraries can be performed by a variety of
techniques. cDNA library construction techniques can be found in
Sambrook, J., et al., supra. It is apparent that DNA encoding HPV18
may also be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by a variety
of techniques. Genomic DNA library construction techniques can be
found in Sambrook, J., et aL supra.
The cloned HPV18 DNA or fragments thereof obtained through the
methods described herein may be recombinantly expressed by
molecular cloning into an expression vector containing a suitable
promoter, and other appropriate transcription regulatory elements,
and transferred into prokaryotic or eukaryotic host cells to
produce recombinant HPV 18. Techniques for such manipulations are
fully described in Sambrook, J., et aL, supra, and are known in the
art.
Expression vectors are defined herein as DNA sequences that are
required for the transcription of cloned copies of genes and the
translation of their mRNAs in an appropriate host. Such vectors can
be used to express eukaryotic genes in a variety of hosts such as
bacteria, bluegreen algae, plant cells, insect cells, fungal cells
and animal cells. Specifically designed vectors allow the shuttling
of DNA between hosts such as bacteria-yeast or bacteria-animal
cells or bacteria-fungal cells or bacteria-invertebrate cells. An
appropriately constructed expression vector should contain: an
origin of replication for autonomous replication in host cells,
selectable markers, a limited number of useful restriction enzyme
sites, a potential for high copy number, and active promoters. A
promoter is defined as a DNA sequence that directs RNA polymerase
to bind to DNA and initiate RNA synthesis. A strong promoter is one
which causes mRNAs to be initiated at high frequency. Expression
vectors may include, but are not limited to, cloning vectors,
modified cloning vectors, specifically designed plasmids or
viruses.
A variety of mammalian expression vectors may be used to express
HPV 18 DNA or fragments thereof in mammalian cells. Commercially
available mammalian expression vectors which may be suitable for
recombinant HPV18 expression, include but are not limited to,
pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTI (Stratagene), pSG5
(Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110),
pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo
(ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and
.lambda.ZD35 (ATCC. 37565).
A variety of bacterial expression vectors may be used to express
HPV18 DNA or fragments thereof in bacterial cells. Commercially
available bacterial expression vectors which may be suitable
include, but are not limited to pET11a (Novagen), lambda gt11
(Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express
HPV 18 or fragments thereof in fungal cells. Commercially available
fungal cell expression vectors which may be suitable include but
are not limited to pYES2 (Invitrogen), Pichia expression vector
(Invitrogen), and Hansenula expression (Rhein Biotech, Dusseldorf,
Germany).
A variety of insect cell expression vectors may be used to express
HPV 18 DNA or fragments thereof in insect cells. Commercially
available insect cell expression vectors which may be suitable
include but are not limited to pBlue Bac III (Invitrogen) and
pAcUW51 (PharMingen, Inc.).
An expression vector containing DNA encoding HPV18 or fragments
thereof may be used for expression of HPV18 proteins or fragments
of HPV18 proteins in a cell, tissues, organs, or animals (including
humans). Host cells may be prokaryotic or eukaryotic, including but
not limited to bacteria such as E. coli, fungal cells such as
yeast, mammalian cells including but not limited to cell lines of
human, bovine, porcine, monkey and rodent origin, and insect cells
including but not limited to Drosophila and silkworm derived cell
lines. Cell lines derived from mammalian species which may be
suitable and which are commercially available, include but are not
limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL
1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),
COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),
3T3 (ATCC CCL 92), NIH/3T3 (ATCC CR L 1658), HeLa (ATCC CCL 2),
C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL
171).
The expression vector may be introduced into host cells via any one
of a number of techniques including but not limited to
transformation, transfection, lipofection, protoplast fusion, and
electroporation. The expression vector-containing cells are
clonally propagated and individually analyzed to determine whether
they produce HPV18 protein. Identification of HPV18 expressing host
cell clones may be done by several means, including but not limited
to immunological reactivity with anti-HPV18 antibodies, and the
presence of host cell-associated HPV 18 activity, such as
HPV18-specific ligand binding or signal transduction defined as a
response mediated by the interaction of HPV 18-specific ligands
with the expressed HPV18 proteins.
Expression of HPV DNA fragments may also be performed using in
vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or
mRNA isolated from HPV18 producing cells can be efficiently
translated in various cell-free systems, including but not limited
to wheat germ extracts and reticulocyte extracts, as well as
efficiently translated in cell based systems, including but not
limited to microinjection into frog oocytes, with microinjection
into frog oocytes being preferred.
Following expression of HPV8 protein(s) in a host cell, HPV18
protein may be recovered to provide HPV18 in purified form. Several
HPV18 purification procedures are available and suitable for use.
As described herein, recombinant HPV 18 protein may be purified
from cell lysates and extracts by various combinations of, or
individual application of salt fractionation, ion exchange
chromatography, size exclusion chromatography, hydroxylapatite
adsorption chromatography and hydrophobic interaction
chromatography.
In addition, recombinant HPV18 may be separated from other cellular
proteins by use of an immunoaffinity column made with monoclonal or
polyclonal antibodies specific for full length nascent HPV18, or
polypeptide fragments ofHPV18. Monoclonal and polyclonal antibodies
may be prepared according to a variety of methods known in the art.
Monoclonal or monospecific antibody as used herein is defined as a
single antibody species or multiple antibody species with
homogenous binding characteristics for HPV18. Homogenous binding as
used herein refers to the ability of the antibody species to bind
to a specific antigen or epitope.
It is apparent that the methods for producing monospecific
antibodies may be utilized to produce antibodies specific for HPV18
polypeptide fragments, or full-length nascent HPV18 polypeptide.
Specifically, it is apparent that monospecific antibodies may be
generated which are specific for the fully functional HPV18 or
fragments thereof.
The present invention is also directed toward methods for screening
for compounds which modulate the expression of DNA or RNA encoding
HPV 18 as well as the function(s) of HPV18 protein(s) in vivo.
Compounds which modulate these activities may be DNA, RNA,
peptides, proteins, or non-proteinaceous organic molecules.
Compounds may modulate by increasing or attenuating the expression
of DNA or RNA encoding HPV18, or the function of HPV18 protein.
Compounds that modulate the expression of DNA or RNA encoding HPV18
or the function ofHPV18 protein may be detected by a variety of
assays. The assay may be a simple "yes/no" assay to determine
whether there is a change in expression or function. The assay may
be made quantitative by comparing the expression or function of a
test sample with the levels of expression or flnction in a standard
sample.
Kits containing HPV18 DNA, fragments of HPV18 DNA, antibodies to
HPV18 DNA or HPV18 protein, HPV18 RNA or HPV18 protein may be
prepared. Such kits are used to detect DNA which hybridizes to
HPV18 DNA or to detect the presence of HPV 18 protein(s) or peptide
fragments in a sample. Such characterization is useful for a
variety of purposes including but not limited to forensic analyses
and epidemiological studies.
Nucleotide sequences that are complementary to the HPV18 encoding
DNA sequence may be synthesized for antisense therapy. These
antisense molecules may be DNA, stable derivatives of DNA such as
phosphorothioates or methylphosphonates, RNA, stable derivatives of
RNA such as 2'-O-alkylRNA, or other HPV18 antisense of
oligonucleotide mimetics. HPV18 antisense molecules may be
introduced into cells by microinjection, liposome encapsulation or
by expression from vectors harboring the antisense sequence. HPV18
antisense therapy may be particularly useful for the treatment of
diseases where it is beneficial to reduce HPV18 activity.
The term "chemical derivative" describes a molecule that contains
additional chemical moieties which are not normally a part of the
base molecule. Such moieties may improve the solubility, half-life,
absorption, etc. of the base molecule. Alternatively the moieties
may attenuate undesirable side effects of the base molecule or
decrease the toxicity of the base molecule. Examples of such
moieties are described in a variety of texts, such as Remington's
Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may
be used alone at appropriate dosages defined by routine testing in
order to obtain optimal inhibition of the HPV18 or its activity
while minimizing any potential toxicity. In addition,
co-administration or sequential administration of other agents may
be desirable.
Advantageously, compounds of the present invention may be
administered in a single daily dose, or the total daily dosage may
be administered in several divided doses. Furthermore, compounds
for the present invention may be administered via a variety of
routes including but not limited to intranasally, orally,
transdermally or by suppository.
For combination treatment with more than one active agent, where
the active agents are in separate dosage formulations, the active
agents can be administered concurrently, or they each can be
administered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention
is selected in accordance with a variety of factors including type,
species, age, weight, sex and medical condition of the patient; the
severity of the condition to be treated; the route of
administration; the renal and hepatic function of the patient; and
the particular compound thereof employed. A physician of ordinary
skill can readily determine and prescribe the effective amount of
the drug required to prevent, counter or arrest the progress of the
condition. Optimal precision in achieving concentrations of drug
within the range that yields efficacy without toxicity requires a
regimen based on the kinetics of the drug's availability to target
sites. This involves a consideration of the distribution,
equilibrium, and elimination of a drug.
In the methods of the present invention, the compounds herein
described in detail can form the active ingredient, and are
typically administered in admixture with suitable pharmaceutical
diluents, excipients or carriers (collectively referred to herein
as "carrier" materials) suitably selected with respect to the
intended form of administration, that is, oral tablets, capsules,
elixirs, syrup, suppositories, gels and the like, and consistent
with conventional pharmaceutical practices.
For instance, for oral administration in the form of a tablet or
capsule, the active drug component can be combined with an oral,
non-toxic pharmaceutically acceptable inert carrier such as
ethanol, glycerol, water and the like. Moreover, when desired or
necessary, suitable binders, lubricants, disintegrating agents and
coloring agents can also be incorporated into the mixture. Suitable
binders include without limitation, starch, gelatin, natural sugars
such as glucose or beta-lactose, corn sweeteners, natural and
synthetic gums such as acacia, tragacanth or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limitation,
sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include, without limitation, starch, methyl
cellulose, agar, bentonite, xanthan gum and the like.
For liquid forms the active drug component can be combined in
suitably flavored suspending or dispersing agents such as the
synthetic and natural gums, for example, tragacanth, acacia,
methyl-cellulose and the like. Other dispersing agents which may be
employed include glycerin and the like. For parenteral
administration, sterile suspensions and solutions are desired.
Isotonic preparations which generally contain suitable
preservatives are employed when intravenous administration is
desired.
Topical preparations containing the active drug component can be
admixed with a variety of carrier materials well known in the art,
such as, e.g., alcohols, aloe vera gel, allantoin, glycerine,
vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and
the like, to form, e.g., alcoholic solutions, topical cleansers,
cleansing creams, skin gels, skin lotions, and shampoos in cream or
gel formulations.
The compounds of the present invention can also be administered in
the form of liposome delivery systems, such as small unilamellar
vesicles, large unilamellar vesicles and multilamellar vesicles.
Liposomes can be formed from a variety of phospholipids, such as
cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be delivered by the use
of monoclonal antibodies as individual carriers to which the
compound molecules are coupled. The compounds of the present
invention may also be coupled with soluble polymers as targetable
drug carriers. Such polymers can include polyvinyl-pyrrolidone,
pyran copolymer, polyhydroxypropylmethacryl-amidephenol,
polyhydroxy-ethylaspartamidephenol, or
polyethyl-eneoxidepolylysine, substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled
to a class of biodegradable polymers useful in achieving,
controlled release of a chug, for example, polylactic acid,
polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthocsters, polyacetals, polydihydro- pyrans,
polycyanoacrylates and cross-linked or a amphipathic block
copolymers of hydrogels.
The following examples illustrate the present invention without,
however, limiting the same thereto.
EXAMPLE 1
Cloning of HPV 18 Genome
Total genomic DNA was prepared from the human cervical
carcinoma-derived cell line, SW756 (Freedman, R. S., et al., 1982,
In Vitro, Vol 18, pages 719-726) by standard techniques. The DNA
was digested with EcoRI and electrophoresed through a 0.8%
low-melting temperature, agarose preparative gel. A gel slice was
excised corresponding to DNA fragments approximately 12 kbp in
length. The agarose was digested using Agarase.TM. enzyme
(Boehringer Mannheim, Inc.) and the size-fractionated DNA was
precipitated, dephosphorylated and ligated with EcoR1 digested
lambda EMBL4 arms (Stratagene, Inc.). The lambda library was
packaged using Gigapack II Gold packaging extract (Stratagene,
Inc), HPV 18-positive clones were identified using a 700 bp, HPV18
L1 DNA probe that was generated by polymerase chain reaction (PCR)
using SW756 DNA as template and oligonucleotide primers that were
designed based on the published HPVl 8 L1 DNA sequence (Cole and
Danos, 1987, J. Mol. Biol., Vol. 193:599-608; Genbank Accession
#X05015). A HPV18-positive, lambda clone was isolated that
contained a 12 kbp EcoRI fragment insert and was designated as
#187-1.
EXAMPLE 2
Construction of Yeast Expression Vectors
The HPV 18 L1 open reading flame (ORF) was amplified by PCR using
clone #187-1 as template, Vent polymrerase.TM. (New England
Biolabs, Inc.), 10 cycles of amplification (94.degree. C., 1 min;
50.degree. C., 1 min; 72.degree. C., 2 min) and the following
oligonucleotide primers which contain flanking BglII sites
(underlined): sense primer,
5'-GAAGATCTCACAAAACAAAATGGCTTTGTGGCGGCCTAGTG-3' (SEQ ID NO:5)
antisense primer,
5'-GAAGATCTTTACTTCCTGGCACGTACACGCACACGC-3' (SEQ ID NO:6).
The sense primer introduces a yeast non-translated leader sequence
(Kniskem, et al., 1986, Gene, Vol. 46:135-141) immediately upstream
to the HPV18 L1 initiating methionine codon (highlighted in bold
print). The 1.5 kbp L1 PCR product was digested with BglII and gel
purified.
The pGAL1-10 yeast expression vector was constructed by isolating a
1.4 kbp SphI fragment from a pUC 18/bidirectional GAL promoter
plasmid which contains the Saccharomyces cerevisiae divergent
GAL1-GAL10 promoters from the plasmid pBM272 (provided by Mark
Johnston, Washington University, St. Louis, Mo.). The divergent
promoters are flanked on each side by a copy of the yeast ADH1
transcriptional terminator, a BamHI cloning site located between
the GAL1 promoter and the first copy of the ADH1 transcriptional
terminator and a SmaI cloning site located between the GALIO
promoter and the second copy of ADH1 transcriptional terminator. A
yeast shuttle vector consisting of pBR322, the yeast LEU2d gene,
and the yeast 2u plasmid (gift of Benjamin Hall, University of
Washington, Seattle, Wash.) was digested with SphI and ligated with
the 1.4 kbp SphI divergent GAL promoter fragment resulting in
pGAL1-10. pGAL1-10 was linearized with BamHI which cuts between the
GAL1 promoter and the ADH1 transcription terminator. The BamHI
digested vector and the BglII digested HPV18 L1 PCR fragment were
ligated and used to transform E. coli DH5 cells (Gibco BRL, Inc.).
A PGAL1-10 plasmid was isolated which contains the HPV18 L1 gene
and was designated p 191-6.
A yeast expression vector that co-expresses both the HPV18 L1 and
L2 genes was constructed. Plasmid p191-6 (pGAL1-10 +HPV18 L1) was
digested with SmaI which cuts between the GAL10 promoter and the
ADH1 transcription terminator. The 1.4 kbp HPV 18 L2 gene was
amplified by PCR as described above using the following
oligonucleotide primers which contain flanking Smal sites
(underlined): sense primer,
5'-TCCCCCGGGCACAAAACAAAATG
GTATCCCACCGTGCCGCACGAC-3' (SEQ ID NO:7),
antisense primer,
5'-TCCCCCGGGCTAGGCCGCCACAAAGCCATCTGC-3'. (SEQ ID NO:8) The sense
primer introduces a yeast non-translated leader sequence (Kniskern
et aL, 1986, supra) immediately upstream to the HPV18 L2 initiating
methionine codon (highlighted in bold print). The PCR fragment was
digested with SmaI, gel purified and ligated with the SmaI digested
p191-6 plasmid. A pGAL1-10 plasmid containing both the HPV18 L1 and
L2 genes was isolated and designated, p195-11.
EXAMPLE 3
Typing of Clinical Samples
Cervical biopsy samples were collected at the Veterans
Administration Medical Center in Indianapolis, IN (courtesy of Dr.
Darron Brown) and at the Albert Einstein Medical Center in
Philadelphia, PA (courtesy of Dr. Joan Adler) and were frozen at
-20.degree. C. DNA was isolated as described by Brown et al., 1993
(Brown, D. et al, 1993, J. Clin. Microbiol., Vol. 31:2667-2673).
Briefly, clinical specimens were processed with a Braun
mikro-dismembrator II (B. Braun Instruments, Melsungen, Germany)
and solubilized in buffer containing 10 mM EDTA and 0.6% (w/v)
sodium dodecyl sulfate (SDS). Samples were adjusted to 20 mM Tris
pH 7.4 and protein was digested with 50 mcg/m I, Proteinase K in
the, presence of 0.1 mcg/mL RNase A followed by extraction with
phenol/chloroform/isoamyl alcohol. DNA was ethanol precipitated and
quantified by spectrophotometry.
The DNA samples were screened for the presence of HPV18 by PCR and
Southern blot analyses. A 256 bp segment of the HPV 18 L1 ORF was
amplified by PCR using the following oligonucleotide primers:
sense primer,
5'-CAATCCTTATATATTAAAGGCACAGGTATG-3', (SEQ ID NO:9) antisense
primer,
5'-CATCATATTGCCCAGGTACAGGAGACTGTG-3'. (SEQ ID NO: 10) The PCR
conditions were according to the manufacturer's recommendations for
the AmpliTaq.TM. DNA Polymerase/GeneAmp.TM. kit (Perkin Elmer
Corp.) except that 0.5 .beta.l of clinical sample DNA was used as
template and 10 pmoles of each primer, 2 mM dNTPs and 2.0 MM
MgCl.sub.2 were in the final reaction mixture. A 2 min, 94.degree.
C. denaturation step was followed by 40 cycles of amplification
(94.degree. C., 1 min; 45.degree. C., 1 min; 72.degree. C., 1
min).
The PCR products were electrophoresed through a 3.0%, agarose gel,
blotted onto nylon membranes and hybridized with a .sup.32
P-labeled HPV18 L1-specific oligonucleotide probe.
EXAMPLE 4
DNA Sequencing of L1 and L2 Genes
The HPV18 L1 and L2 genes in clones #187-1, p191-6 and p195-11 were
sequenced using the PRIZM Sequencing kit and the automated DNA ABI
Sequencer #373A (Applied Biosystems). To obtain a consensus HPV18
sequence, portions of the L1 gene DNA were amplified by PCR from
human clinical isolates, sequenced and compared to the claimed and
published sequences. A 256 bp fragment (nucleotides 817-1072) was
amplified from each clinical DNA isolate for this purpose using the
oligonucleotides and heating cycles described in Example 3. The
following primers,
5'-GAAGATCTCACAAAACAAAAATGGCTTTGTGGCGGCCTAGTG-3' (SEQ ID NO:11)
and
5'-CCTAACGTCCTCAGAAACATTAGAC-3' (SEQ ID NO: 12) were used to
amplify an amino-terminal 432 bp portion of L1 DNA (nucleotides
1-431) using the heating cycles described in Example 3. Both PCR
products were ligated separately with plasmid pCRII (Invitrogen
Corp.) using the reagents and procedures recommended by the
manufacturer. Plasmid DNA was isolated from the transformants and
those containing EcoRI inserts were sequenced.
EXAMPLE 5
Analysis of DNA and Deduced Amino Acid Sequences
The nucleotide and deduced amino acid (aa) sequences of the claimed
HPV 18 L1 are shown in FIG. 1. The DNA sequence was derived from a
consensus of clones #187-1, p 191-6 and p 195-11. A comparison of
the claimed HPV18 L1 nucleotide sequence with the published HPV 18
L1 sequence (Genbank Accession #X05015) identified 20 bp changes
out of 1524 bps. Five of the nucleotide changes (C to G at position
89, C to A at 263, C to G at 848, G to A at 967 and C to G at 1013)
result in amino acid substitutions. The five residue differences
from published are P to R at aa positions 30, 283 and 338, T to N
at aa 88 and V to I at aa 323 (FIG. 2). Positions 88 and 323
represent conservative changes while the three P to R changes may
substantially alter the physical properties of the expressed L1
protein.
A comparison of the amino acid sequences derived from clinical
isolates (numbers 354, 556, 755, 697, 795 and 23) with the claimed
sequence and the published sequence is shown in FIG. 2. There are
four locations where the clinical isolates and the claimed sequence
differ from the published sequence. Positions 30, 283 and 338
encode arginine (R) in all the isolates found to date, including
the claimed sequence. This is in sharp contrast to the published
sequence which has prolines (P) at each of these locations.
Furthermore, position 88 is an asparagine (N) in the isolates and
the claimed sequence but is a threonine (T) in the published
sequence. The last difference, position 323, was found to be a
valine (V) in many of the clinical isolates and the published
strain verses an isoleucine (I) in the claimed sequence and one of
file isolates (#23). The conclusion is that the claimed sequence
reflects the predominant viral sequences that are associated with
clinical infections and the absence of isolates containing any of
the position 30, 283 or 338 prolines of the published sequence
suggests that the published clone is either an artefact or an
inconsequential subtype.
The nucleotide and deduced aa sequences of HPV18 L2 were derived
from a consensus sequence of clones #187-1 and p195-11 and are
shown in FIG. 3. A comparison of the L2 nucleotide sequence with
the published HPV18 sequence (Genbank Accession #X05015) identified
40 bp changes out of 1389 bps. The bp differences result in 14
changes at the aa level: P to S at aa 29, P to N at aa 33, A to Sat
aa 177, D to Eat aa 266, D to N at aa 270, D to G at aa 346, M to I
at 355, V to M at aa 359, S to P at aa 365, F to S at aa 369, F to
V at aa 371, F to S at aa 372, K to T at aa 373 and S to P at aa
409.
EXAMPLE 6
Generation of HPV 18 L2 Antiserum
HPV18 L2 specific antibodies were prepared in goats using a
trpE-HPV 18 L2 fusion protein expressed in E. coli. The full-length
L2 ORF was amplified by PCR using oligonucleotide primers providing
HindIlI and Bam HI sites flanking the 5'- and 3'-ends,
respectively. The L2 fragment was inserted into the HindIII-BamHI
digested, pATH23 expression plasmid (Koerner et al., 1991, Meth.
Enzymol. Vol. 194:477-490). The fusion protein was expressed in E.
coli RR1 cells (Gibco BRL, Inc.) after induction with
3-b-indoleacrylic acid. The insoluble fraction was analyzed by
SDS-PAGE followed by staining with Coomassie Blue. The trpE-L2
fusion protein accounted for the major portion of the E. coli
insoluble fraction. Goats were immunized with the trpE-L2 fusion
protein according to the standard protocol of Pocono Rabbit Farm
and Laboratory, Inc. for fusion protein antigens (Protein Rabbit
Farm, Canadensis, Pa.).
EXAMPLE 7
Preparation of Yeast Strain U9
Saccharomyces cerevisiae strain 2150-2-3 (MATalpha, leu2-04, adel,
cir.degree.) was obtained from Dr. Leland Hartwell (University of
Washington, Seattle, Wash). Cells of strain 2150-2-3 were
propagated overnight at 30.degree. C. in 5 mL, of YEHD medium
(Carty et al, J Ind Micro2 (1987) 117-121). The cells were washed 3
times in sterile, distilled water, resuspended in 2 ml of sterile
distilled water, and 0.1 mL of cell suspension was plated onto each
of six 5 fluoro-orolic acid (FOA) plates in order to select for
ura3 mutants (Cold Spring Harbor Laboratory Manual for Yeast
Genetics). The plates were incubated at 30.degree. C. The medium
contained per 250 mL, distilled water: 3.5 g, Difco Yeast Nitrogen
Base without amino acids and ammonium sulfate; 0.5 g
5-Fluoro-orotic acid; 25 mg Uracil; and 10.0 g Dextrose.
The medium was sterilized by filtration through 0.2 .mu.m membranes
and then mixed with 250 mL of 4% Bacto-Agar (Difco) maintained at
50.degree. C., 10 mL of a 1.2 mg/mL solution of adenine, and 5 mL
of L-leucine solution (180 mg/50 mL). The resulting medium was
dispensed at 20 mL per petri dish.
After 5 days of incubation, numerous colonies had appeared. Single
colonies were isolated by restreaking colonies from the initial FOA
plates onto fresh FOA plates which were then incubated at
30.degree. C. A number of colonies from the second set of FOA
plates were tested for the presence of the ura3 mutation by
replica-plating onto both YEHD plates and uracil-minus plates. The
desired result was good growth on YEHD and no growth on
uracil-minus medium. One isolate (U9) was obtained which showed
these properties. It was stored as a frozen glycerol stock (strain
#325) at -70.degree. C. for later use.
EXAMPLE 8
Preparation of a Vector for Disruption of the Yeast MNN9 Gene
In order to prepare a vector for disruption of the MAN9 gene, it
was necessary to first clone the MNN9 gene from S. cerevisiae
genomic DNA. This was accomplished by standard Polymerase Chain
Reaction (PCR) technology. A 5' sense primer and 3' antisense
primer for PCR of the full length MNN9 coding sequence were
designed based on the published sequence for the yeast MNN9 gene
(Zymogenetics: EPO Patent Application No. 88117834.7, Publication
No. 0-314-096-A2). The following oligodeoxynucleotide primers
containing flanking HindIlI sites (underlined) were used:
sense primer: 5'-CTT AAA GCT TAT GTC ACT TTC TCT TGT ATC G-3' (SEQ
ID NO:13)
antisense primer: 5'-TGA TAA GCT TCA ATG GTT CTC TTC CTC-3' (SEQ ID
NO:14).
The initiating methionine codon for the MNN9 gene is highlighted in
bold print. The PCR was conducted using genomic DNA from S.
cerevisiae strain JRY 188 as template, Taq DNA polymrerase (Perkin
Elmer) and 2.5 cycles of amplification (94.degree. C. 1 min.,
37.degree. C. 2 min., 72.degree. C. 3 min.). The resulting 1.2 kbp
PCR fragment was digested with HindIII, gel-purified, and ligated
with HindIII-digested, alkaline-phosphatase treated pUC13
(Pharmacia). The resulting plasmid was designated p1183.
In order to disrupt the MNN9 gene with the yeast URA3 gene, the
plasmid pBR322-URA3 (which contains the 1.1 Kbp HindIII fragment
encoding the S. cerevisiae URA3, gene subcloned into the HindIII
site of pBR322) was digested with HindIII and the 1.1 kbp DNA
fragment bearing the functional UWA3 gene was gel-purified, made
blunt-ended with T4 DNA polymerase, and then ligated with
PmlI-digested plasmid p1183 (PmlI cuts within the MNN9 coding
sequence). The resulting plasmid p1199 contains a disruption of the
MNN9 gene by the functional URA3 gene.
EXAMPLE 9
Construction of U9-derivative Strain 1372 Containing Disruption of
MNN9 Gene
For disruption of the MNN9 gene in strain U9 (#325), 30 .mu.g of
plasmid p1199 were digested with HindIII to create a linear mnn9::
URA3 disruption cassette. Cells of strain 325 were transformed with
the HindIII-digested p1199 DNA by the spheroplast method (Hinnen et
al., 1978, Proc. Natl. Acad. Sci. USA 75:1929-1933) and
transformants were selected on a synthetic agar medium lacking
uracil and containing 1.0 M sorbitol. The synthetic medium
contained, per liter of distilled water: Agar, 20 g; Yeast nitrogen
base w/o amino acids, 6.7 g; Adenine, 0.04 g; L-tyrosine, 0.05 g;
Sorbitol, 182 g; Glucose, 20 g; and Leucine Minus Solution #2, 10
ml. Leucine Minus Solution #2 contains per liter of distilled
water: L-arginine, 2 g; L histidine, 1 g; L-Leucine, 6 g;
L-Isoleucine, 6 g; L-lysine, 4 g; L-methionine, 1 g;
L-phenylalanine, 6 g; L-thrconine, 6 g; L-tryptophan, 4 g.
The plates were incubated at 30.degree. C. for five days at which
time numerous colonies had appeared. Chromosomal DNA preparations
were made from 10 colonies and then digested with EcoRI plus
HindIII. The DNA digests were then evaluated by Southern blots (J.
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
edition, Cold Spring Harbor Laboratory Press, 1989) using the 1.2
kbp HindIII fragment bearing the MNN9 gene (isolated from plasmid
p1199) as a probe. An isolate was identified (strain #1372) which
showed the expected DNA band shifts on the Southern blot as well as
the extreme clumpiness typically shown by mnn9 mutants.
EXAMPLE 10
Constriction of a Vector for Disruption of Yeast HIS3 Gene
In order to construct a disruption cassette in which the S.
cerevisiae HIS3 gene is disrupted by the URA3 gene, the plasmid
YEp6 (K. Struhl et al., 1979, Proc. Natl. Acad. Sci USA 76:1035)
was digested with BamHI and the 1.7 kbp BamHI fragment bearing the
HIS3 gene was gel-purified, made blunt-ended with T4 DNA
polymerase, and ligated with pUC 18 which has been previously
digested with BamHI and treated with T4 DNA polymerase. The
resulting plasmid (designated p1501 or pUC18-HIS3) was digested
with Nhel (which cuts in the HIS3 coding sequence), and the vector
fragment was gel-purified, made blunt-ended with T4 DNA polymerase,
and then treated with calf intestine alkaline phosphatase. The URA3
gene was isolated from the plasmid pBR322-URA3 by digestion with
HindIII and the 1.1 kbp fragment bearing the URA3 gene was
gel-purified, made blunt-ended with T4 DNA polymerase, and ligated
with the above pUC18-HIS3 Nhel fragment. The resulting plasmid
(designated pUC18-his3::URA3 or p1505) contains a disruption
cassette in which the yeast HIS3 gene is disrupted by the
finctional URA3 gene.
EXAMPLE 11
Construction of Vector for Disruption of Yeast PRB1 Gene by the
HIS3 Gene
Plasmid FP8AH bearing the S. cerevisiae PRB1 gene was provided by
Dr. E. Jones of Carnegie-Mellon Univ. (C.M. Moehl et al., 1987,
Genetics 115:255-263). It was digested with HindIII plus XhoI and
the 3.2 kbp DNA fragment bearing the PRBI gene was gel-purified and
made blunt-ended by treatment with T4 DNA polymerase. The plasmid
pUC18 was digested with BamHI, gel-purified and made blunt-ended by
treatment with T4 DNA polymerase. The resulting vector fragment was
ligated with the above PRB1 gene fragment to yield the plasmid
pUC18 PRB1. Plasmid YEp6, which contains the HIS3 gene, was
digested with BamHI. The resulting 1.7 kbp BamHI fragment bearing
the functional HIS3 gene was gel-purified and then made blunt-ended
by treatment with T4 DNA polymerase. Plasmid pUC18-PRB1 was
digested with EcoRV plus NcoI which cut within the PRBI coding
sequence and removes the protease B active site and flanking
sequence. The 5.7 kbp EcoRV-NcoI fragment bearing the residual 5'
and 3'-portions of the PRBI coding sequence in pUC18 was
gel-purified, made blunt-ended by treatment with T4 DNA polymerase,
dephosphorylated with calf intestine alkaline phosphatase, and
ligated with the blunt-ended HIS1 fragment described above. The
resulting plasmid (designated pUC18-prb1::HIS3, stock #1245)
contains the functional HIS3 gene in place of the portion of the
PRB1 gene which has been deleted above.
EXAMPLE 12
Construction of a U9-related Yeast Strain Containing Disruptions of
Both the MNN9 and PRB 1 Genes
The U9-related strain 1372 which contains a MNN9 gene disruption
was described in Example 9. Clonal isolated of strain 1372 were
passaged on FOA plates (as described in Example 7) to select ura3
mutants. A number of ura3 isolates of strain 1372 were obtained and
one particular isolate (strain 12930-190-S1-1) was selected for
subsequent disruption of the HIS3 gene. The pUC18-his3::URA3 gene
disruption vector (p1505) was digested with XbaI plus EcoRI to
generate a linear his3::URA3 disruption cassette and used for
transformation of strain 12930-190-S1-1 by the lithium acetate
method [Methods in Enzymology, 194:290 (1991)]. URA+ transformants
were selected on synthetic agar medium lacking uracil, restreaked
for clonal isolates on the same medium, and then replica-plated
onto medium lacking either uracil or histidine to screen for those
isolates that were both Ura+ and His-. One isolate (strain
12930-230-1) was selected for subsequent disruption of the PRB1
gene. The PRB1 gene disruption vector (pUCI 8-prb 1::HIS3, stock
#1245) was digested with SacI plus XbaI to generate a linear
prbl::HIS3 disruption cassette and used for transformation of
strain 12930-230-1 by the lithium acetate method. His+
transformants were selected on agar medium lacking histidine and
restreaking on the same medium for clonal isolates. Genomic DNA was
prepared from a number of the resulting His+ isolates, digested
with EcoRI, and then electrophoresed on 0.8% agarose gels. Southern
blot analyses were then performed using a radio-labeled 617 bp
probe for the PRB1 gene which has been prepared by PCR using the
following oligodeoxynucleotide primers:
5' TGG TCA TCC CAA ATC TTG AAA 3' SEQ ID NO:15)
5.degree. CAC CGT AGT GTT TGG AAG CGA 3' SEQ ID NO:16)
Eleven isolates were obtained which showed the expected
hybridization of the probe with a 2.44 kbp prb1::HIS3 DNA fragment.
This was in contrast to hybridization of the probe with the 1.59
kbp fragment for the wild-type PRB1 gene. One of these isolates
containing the desired prb1::HIS3 disruption was selected for
further use and was designated strain #1558.
EXAMPLE 13
Expression of HPV 18 L1 and L2 in Yeast
Plasmids p191-6 (pGAL1-10+HPV 18 L1) and p195-11 (pGAL1-10+HPV18
L1+L2) were used to transform S. cerevisiae strain #1558 (MATa,
leu2-04m prbI::HIS3, mnn9::URA3, adel, cir.degree.). Clonal
isolates were grown at 30.degree. C. in YEHD medium containing 2%
galactose for 88 hours. After harvesting the cells, the cell
pellets were broken with glass beads and cell lysates analyzed for
the expression of HPV18 L1 and/or HPV18 L2 protein by immunoblot
analysis. Sarnples containing 25.multidot..mu.g of total cellular
protein were electrophoresed through 10% Tris-Glycine gels (Novex,
Inc.) under denaturing conditions and electroblotted onto
nitrocellulose filters. L1 protein was immunodetected using rabbit
antiserum raised against a trpE-HPV1 L1 fusion protein as primary
antibody (Brown et al., 1994, Virology 201:46-54) and horseradish
peroxidase (HRP)-linked donkey anti-rabbit IgG (Amersham, Inc.) as
secondary antibody. The filters were processed using the
chemiluminescent ECL.TM. Detection Kit (Amersham, Inc.). A 50-55
KDa L1 protein band was detected in both the L1 and L1+L2
coexpressor yeast clones (strains 1725 and 1727, respectively) and
not in the negative control (PGAL1-10 without L1 or L2 genes) (FIG.
4).
The HPV18 L2 protein was detected by Western analysis using goat
polyclonal antiserum raised against a trpE-HPV18 L2 fusion protein
as primary antibody followed by HRP-conjugated, rabbit anti-goat
IgG (Kirkegaard and Perry Laboratories, Gaithersburg, Md.). The
filters were processed as described above. The L2 protein was
detected as a 75 kDa protein band in the L1 and L2 coexpressor
yeast clone (strain 1727) but not in either the negative control or
the L1 expresser clone (FIG. 5).
EXAMPLE 14
Fermentation of HPV18 L1 (Strain 1725) and 18 L1+.DELTA.L2 (Strain
1727).
Surface growth of a plate culture of strains 1725 and 1727 was
aseptically transferred to a leucine-free liquid medium containing
(per L): 8.5 g Difco yeast nitrogen base without amino acids and
ammonium sulfate; 0.2 g adenine; 0.2 g uracil; 10 g succinic acid;
5 g ammonium sulfate; 40 g glucose; 0.25 g L-tyrosine; 0.1
L-arginine; 0.3 g L-isoleucine; 0.05 g L-methionine; 0.2 g
L-tryptophan; 0.05 g L-histadine; 0.2 g L-lysine; 0.3 g
L-phenylalanine; this medium was adjusted to pH 5.0-5.3 with NaOH
prior to sterilization. After growth at 28.degree. C., 250 rpm on a
rotary shaker, frozen culture vials were prepared by adding sterile
glycerol to a final concentration of 17% (w/v) prior to storage at
-70.degree. C. (1 mL per cryovial). Inocula were developed in the
same medium (500 mL per 2-L flask) and were started by transferring
the thawed contents of a frozen culture vial and incubating at
28.degree. C., 250 rpm on a rotary shaker for 29 hr. Fermentations
of each strain used a New Brunswick SF-116 fermentor with a working
volume of 10 L after inoculation. The production medium contained
(per L): 20 g Difco yeast extract; 10 g Sheffield HySoy peptone, 20
g glucose; 20 g galactose; 0.3 mL Union Carbide UCON LB-625
antifoan; the medium was adjusted to pH 5.3 prior to sterilization.
The entire contents (500 mL) of the 2-L inoculum flask was
transferred to the fermentor which was incubated at 28.degree. C.,
5 L air per min, 400 rpm, 3.5 psi pressure. Agitation was increased
as needed to maintain dissolved oxygen levels of greater than 40%
of saturation. Progress of the fermentation was monitored by
off-line glucose measurements (Beckman Glucose 2 Analyzer) and
on-line mass spectrometry (Perkin-Elmer 1200). After incubation for
66 hr, cell densities of 9.5 to 9.7 g dry cell weight per L were
reached. The cultures were concentrated by hollow fiber filtration
(Amicon H5MP01-43 cartridge in an Amicon DC-10 filtration system)
to ca. 2 L, diafiltered with 2 L phosphate-buffered saline, and
concentrated further (to ca. 1 L) before dispensing into 500-mL
centrifuge bottles. Cell pellets were collected by centrifugation
at 8,000 rpm (Sorval GS-3 rotor) for 20 min at 4.degree. C. Afler
decanting the supernatant, the pellets (total 191 to 208 g wet
cells) were stored at -70.degree. C. until use.
EXAMPLE 15
Purification of Recombinant HPV Type 18 L1 Capsid Proteins
All steps performed at 4.degree. C. unless noted.
Cells were stored frozen at -70.degree. C. Frozen cells (wet
weight=126 g) were thawed at 20-23.degree. C. and resuspended in 70
mL "Breaking Buffer" (20 mM sodium phosphate, pH 7.2, 100 mM NaCl).
The protease inhibitors PMSF and pepstatin A were added to final
concentrations of 2 mM and 1.7 .mu.M, respectively. The cell slurry
was broken at a pressure of approximately 16,000 psi by 4 passes in
a M110-Y Microfluidizer Corp., Newton, Mass.). The broken cell
slurry was centrifuged at 12,000.times.g for 40 min to remove
cellular debris. The supernatant liquid containing L1 antigen was
recovered.
The supematant liquid was diluted 1:5 by addition of Buffer A (20
mM MOPS, pH 7.0) and applied to an anion exchange capture column
(9.0 cm ID.times.3.9 cm) of a Fractogel.RTM. EMD TMAE-650 (M) resin
(EM Separations, Gibbstown, N.J.) equilibrated in Buffer A.
Following a wash with a Buffer A, the antigen was eluted with a
gradient of 0-1.0 M NaCl in Buffer A. Column fractions were assayed
for total protein by the Bradford method. Fractions were then
analyzed at equal total protein loadings by Western blotting and
SDS-PAGE with silver stain detection.
TMAE fractions showing comparable purity and enrichment of L1
protein were pooled. The antigen was concentrated by ammonium
sulfate fractionation. The solution was adjusted to 35% saturated
ammonium sulfate by adding solid reagent while gently stirring over
10 min. The sample was placed on ice and precipitation allowed to
proceed for 4 hours. The sample was centrifuged at 16,000.times.g
for 45 min. The pellet was resuspended in 20.0 mL PBS (6.25 mM Na
phosphate, pH 7.2, 150 mM NaCl.
The resuspended pellet was chromatographed on a size exclusion
column (2.6 cm ID.times.89 cm) of Sephacryl 500 HR resin
(Pharmacia, Piscataway, N.J.). Running buffer was PBS. Fractions
were analyzed by western blotting and SDS-PAGE with silver stain
detection. The purest fractions were pooled. the resulting pool was
concentrated in a 50 mL stirred cell using 43 mm YM-100 flat-sheet
membranes (Amicon, Beverly, Mass.) at a N.sub.2 pressure of 4-6
psi.
Final product was analyzed by western blotting and SDS-PAGE with
colloidal Coomassie detection. The L1 protein was estimated to be
50-60% homogeneous. The identity of L1 protein was confirmed by
western blotting. The final product was aliquoted and stored at
-70.degree. C. The process resulted in a total of 12.5 mg
protein.
Bradford Assay for Total Protein
Total protein was assayed using a commercially available Coomassie
Plus.RTM. kit (Pierce, Rockford, Ill.). Samples were diluted to
appropriate levels in Milli-Q-H.sub.2 O. Volumes required were 0.1
mL and 1.0, L for the standard and microassay protocols,
respectively. For both protocols, BSA (Pierce, Rockford, Ill.) was
used to generate the standard curve. Assay was performed according
to manufacturer's recommendations. Standard curves were plotted
using CricketGraph.RTM. software on a Macintosh IIci computer.
SDS-PAGE and Western Blot Assays
All gels, buffers, and electrophoretic apparatus were obtained from
Novex (San Diego, Calif.) and were run according to manufacturer's
recommendations. Briefly, samples were diluted to equal protein
concentrations in Milli-Q-H.sub.2 O and mixed 1:1 with sample
incubation buffer containing 200 mM DTT. Samples were incubated 15
min at 100.degree. C. and loaded onto pre-cast 12% Tris-glycine
gels. The samples were electrophoresed at 125V for 1 hr 45 nin.
Gels were developed using either silver staining by a variation of
the method of Heukeshoven and Demick [Electrophoresis, 6 (1985)
103-112] or colloidal Coomassie staining using a commercially
obtained kit (Integrated Separation Systems, Natick, Mass.).
For western blots, proteins were transferred to PVDF membranes at
25V for 40 min. Membranes were washed with Milli-Q-H.sub.2 O and
air-dried. Primary antibody was polyclonal rabbit antiserum raised
against a TrpE-HPV11L1 fusion protein (gift or Dr D. Brown).
Previous experiments had shown this antiserum to cross react with
HPV type 18 L1 on western blots. The antibody solution was prepared
by dilution of antiserum in blotting buffer (5% non-fat milk in
6.25 mM Na phosphate, pH 7.2, 150 mM NaCl, 0.2% NaN3). Incubation
was for at least 1 hour at 20-23.degree. C. The blot was washed for
1 min each in three changes of PBS (6.25 mM Na phosphate, pH 7.2,
150 mM NaCl). Secondary antibody solution was prepared by diluting
goat anti-rabbit IgG alkaline phosphatase-linked conjugate
antiserum (Pierce, Rockford, Ill.) in blotting buffer. Incubation
proceeded under the same conditions for at least 1 hour. Blots were
washed as before and detected using a 1 step NBT/BCIP substrate
(Pierce, Rockford, Ill.).
EXAMPLE 16
Electron Microscopic Studies
For EM analysis (Structure Probe, West Chester, Pa.), and aliquot
of each sample was placed on 200-mesh carbon-coated copper grids. A
drop of 2% phosphotungstic acid (PTA), pH 7.0 was placed on the
grid for 20 seconds. The grids were allowed to air dry prior to
transmission EM examination. All microscopy was done using a JEOL
100CX transmission electron microscope (JEOL USA, Inc.) at an
accelerating voltage of 100 kV. The micrographs generated have a
final magnification of 100,000.times.. Virus-like particles were
observed in the 50-55 nm diameter size range in the yeast sample
harboring the HPV18 L1 expression plasmid (FIG. 6). No VLPs were
observed in the yeast control samples.
EXAMPLE 17
Sub-cloning of the HPV18 cDNA into Expression Vectors
The cDNA encoding HPV18 is sub-loned into several vector for
expression of the HPV18 protein in transfected host cells and for
in vitro transcription/translation. These vectors include
pBluescript II SK+ (where expression is driven by T7 or T3
promoters) pcDNA I/Amp (where expression is driven by the
cytomegalovirus (CMV) promoter), pSZ9016-1 (where expression is
driven by the HIV long terminal repeat (LTR) promoter) and the
baculovirus transfer vector pAcUW5 1 (PharMinger, Inc.) (where
expression is driven by he polyhedrin (PH) promoter) for producing
recombinant baculovirus containing the HPV18 encoding DNA
sequence.
a) pBluescript II SK+HPV18. The full length HPV18 cDNA clone is
retrieved from lambda bacteriophage by limited Eco RI digestion and
ligated into Eco RI-cut, CIP-treated pBluescript II SK+. Separate
subclones are recovered in which the sense orientation of HPV18
followed either the T7 or T3 promoters.
b) RcDNA I/Amp:HPV18. To facilitate directional cloning, HPV18 is
excised from a purified plasmid preparation of pBluescript II
SK+:HPV18 in which the HPV18 DNA sequence is downstream of the T7
promoter using Eco RV and Xba I. The resulting downstream of the T7
promoter using Eco RV and Xba I. HPV18 fragment is purified and
ligated into Eco RV-cut, Xba I-cut, CIP-treated pcDNA I/Amp such
that the HPV18 encoding DNA is downstream of the CMV promoter.
c) pSZ9016-1:HPV18. HPV18 is excised from pBluescript II SK+:HPV18
by limited Eco RI digestion and subsequent purification of the 1.3
Kb fragment from agarose gets. The resulting Eco RI HPV18 fragment
is ligated into Eco RI-cut, CIP-treated pSZ9016-1. Subclones are
selected in which the sense orientation of HPV18 is downstream of
the HIV LTR promoter.
d) pAcUW51:HPV18L1. The full-length HPV18 L1 ORF was amplified by
PCR from clone #187-1 using oligonucleotide primers providing
flanking BglII sites. The L1 gene was inserted into the BamHI site
of the baculovirus transfer vector, pAcUW51 (PharMingen, Inc.)
under control of the polyhedrin promoter. Recombinant baculoviruses
were generated containing the HPV18 L1 expression cassette
according to the procedures described in the PharMingen Manual.
Recombinant clones were purified by limiting dilution and dot blot
hybridization.
EXAMPLE 18
Expression of the HPV18 Polypeptide by in Vitro
Transcription/Translation and by Transfection Into Host Cells
Vectors containing HPV DNA sequences are used to drive the
translation of the HPV 18 polypeptide in rabbit reticulocyte
lysates, mammalian host cells, and in baculovirus infected insect
cells. The experimental procedures are essentially those outlined
in the manufacturers' instructions.
a) In vitro Transcription/Translation. pBluescript III SK+:HPV1 8
plasmid DNA (with HPV18 in the T7 orientation) is linearized by Bam
HI digestion downstream of the HPV18 insert. The linearized plasmid
is purified and used as a template for run-off transcription using
T7 RNA polymerase in the presence of m7G(5')ppp(5')G. The resulting
capped HPV18 transcripts are purified by LiCI precipitation and
used to drive the translation of HPV18 in nuclease-pretreated
rabbit reticulocyte lysate in the presence of L-[.sup.35
S]methionine.
b) Expression in Mammalian Cells. The HPV18 protein is expressed in
mammalian host cells following transfection with either pcDNA
I/Amp:HPV18 (under control of the CMV promoter) or pSZ9016-1:HPV18
under control of the HIV ITR promoter in the later case
(pSZ9016-1:HPV18), cells are co-transfected with the TAT expressing
plasmid pSZ9016:TAT. For both HPV18 expression plasmids, COS-7
cells are transfected using either DEAE-dextran or lipofection with
Lipofectamine (BRL).
c) Expression in Insect Cells. The HPV18 L1-containing baculovirus
transfer vector pAcUW5 I :HPV18 L1 is used to produce recombinant
baculovirus (Autographa californica) by in vivo homologous
recombination. Epitope tagged HPV18 L1 is then expressed in Sf9
(Spodoptera frugiperda) insect cells grown in suspension culture
following infection with the HPV18-containing recombinant
baculovirus.
EXAMPLE 19
Compounds that affect HPV18 activity may be detected by a variety
of methods. A method of identifying compounds that affect HPV18
comprises:
a) mixing a test compound with a solution containing HPV18 to form
a mixture;
b) measuring HPV18 activity in the mixture; and
c) comparing the HPV18 in the mixture to a standard.
Compounds that affect HPV18 activity may be formulated into
pharmaceutical compositions. Such pharmaceutical compositions may
be useful for treating diseases or conditions that are
characterized by HPV18 infection.
EXAMPLE 20
DNA which is structurally related to DNA encoding HPV18 is detected
with a probe. A suitable probe may be derived from DNA having all
or a portion of the nucleotide sequence of FIG. 1 or FIG. 3, RNA
encoded by DNA having all or a portion of the nucleotide sequence
of FIG. 1 or FIG. 3 or degenerate oligonucleotides derived from a
portion of the sequence of FIG. 1 or FIG. 3.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 16 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 1524 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: HPV18 L1 Consensus Sequence
<400> SEQUENCE: 1 atggctttgt ggcggcctag tgacaatacc gtataccttc
cacctccttc tgtggcaaga 60 gttgtaaata ctgatgatta tgtgactcgc
acaagcatat tttatcatgc tggcagctct 120 agattattaa ctgttggtaa
tccatatttt agggttcctg caggtggtgg caataagcag 180 gatattccta
aggtttctgc ataccaatat agagtatttc gggtgcagtt acctgaccca 240
aataaatttg gtttacctga taatagtatt tataatcctg aaacacaacg tttagtgtgg
300 gcctgtgctg gagtggaaat tggccgtggt cagcctttag gtgttggcct
tagtgggcat 360 ccattttata ataaattaga tgacactgaa agttcccatg
ccgctacgtc taatgtttct 420 gaggacgtta gggacaatgt gtctgtagat
tataagcaga cacagttatg tattttgggc 480 tgtgcccctg ctattgggga
acactgggct aaaggcactg cttgtaaatc gcgtccttta 540 tcacagggcg
attgcccccc tttagaactt aagaacacag ttttggaaga tggtgatatg 600
gtagatactg gatatggtgc catggacttt agtacattgc aagatactaa atgtgaggta
660 ccattggata tttgtcagtc tatttgtaaa tatcctgatt atttacaaat
gtctgcagat 720 ccttatgggg attccatgtt tttttgctta cgacgtgagc
agctttttgc taggcatttt 780 tggaataggg caggtactat gggtgacact
gtgcctcaat ccttatatat taaaggcaca 840 ggtatgcgtg cttcacctgg
cagctgtgtg tattctccct ctccaagtgg ctctattgtt 900 acctctgact
cccagttgtt taataaacca tattggttac ataaggcaca gggtcataac 960
aatggtatct gctggcataa tcaattattt gttactgtgg tagataccac tcgtagtacc
1020 aatttaacaa tatgtgcttc tacacagtct cctgtacctg ggcaatatga
tgctaccaaa 1080 tttaagcagt atagcagaca tgttgaagaa tatgatttgc
agtttatttt tcagttatgt 1140 actattactt taactgcaga tgttatgtcc
tatattcata gtatgaatag cagtatttta 1200 gaggattgga actttggtgt
tccccccccg ccaactacta gtttggtgga tacatatcgt 1260 tttgtacaat
ctgttgctat tacctgtcaa aaggatgctg caccagctga aaataaggat 1320
ccctatgata agttaaagtt ttggaatgtg gatttaaagg aaaagttttc tttggactta
1380 gatcaatatc cccttggacg taaatttttg gttcaggctg gattgcgtcg
caagcccacc 1440 ataggccctc gtaaacgttc tgctccatct gccactacgt
cttctaaacc tgccaagcgt 1500 gtgcgtgtac gtgccaggaa gtaa 1524
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2
<211> LENGTH: 507 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: HPV18 L1 Consensus Sequence <400> SEQUENCE: 2
Met Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro 1 5
10 15 Ser Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr
Ser 20 25 30 Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val
Gly Asn Pro 35 40 45 Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys
Gln Asp Ile Pro Lys 50 55 60 Val Ser Ala Tyr Gln Tyr Arg Val Phe
Arg Val Gln Leu Pro Asp Pro 65 70 75 80 Asn Lys Phe Gly Leu Pro Asp
Asn Ser Ile Tyr Asn Pro Glu Thr Gln 85 90 95 Arg Leu Val Trp Ala
Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro 100 105 110 Leu Gly Val
Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp 115 120 125 Thr
Glu Ser Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg 130 135
140 Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly
145 150 155 160 Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr
Ala Cys Lys 165 170 175 Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro
Leu Glu Leu Lys Asn 180 185 190 Thr Val Leu Glu Asp Gly Asp Met Val
Asp Thr Gly Tyr Gly Ala Met 195 200 205 Asp Phe Ser Thr Leu Gln Asp
Thr Lys Cys Glu Val Pro Leu Asp Ile 210 215 220 Cys Gln Ser Ile Cys
Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp 225 230 235 240 Pro Tyr
Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe 245 250 255
Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro 260
265 270 Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly
Ser 275 280 285 Cys Val Tyr Ser Pro Ser Pro Ser Gly Ser Ile Val Thr
Ser Asp Ser 290 295 300 Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys
Ala Gln Gly His Asn 305 310 315 320 Asn Gly Ile Cys Trp His Asn Gln
Leu Phe Val Thr Val Val Asp Thr 325 330 335 Thr Arg Ser Thr Asn Leu
Thr Ile Cys Ala Ser Thr Gln Ser Pro Val 340 345 350 Pro Gly Gln Tyr
Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val 355 360 365 Glu Glu
Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu 370 375 380
Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu 385
390 395 400 Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser
Leu Val 405 410 415 Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr
Cys Gln Lys Asp 420 425 430 Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr
Asp Lys Leu Lys Phe Trp 435 440 445 Asn Val Asp Leu Lys Glu Lys Phe
Ser Leu Asp Leu Asp Gln Tyr Pro 450 455 460 Leu Gly Arg Lys Phe Leu
Val Gln Ala Gly Leu Arg Arg Lys Pro Thr 465 470 475 480 Ile Gly Pro
Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys 485 490 495 Pro
Ala Lys Arg Val Arg Val Arg Ala Arg Lys 500 505 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211>
LENGTH: 1389 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: HPV18
L2 Consensus Sequence <400> SEQUENCE: 3 atggtatccc accgtgccgc
acgacgcaaa cgggcttcgg tgactgactt atataaaaca 60 tgtaaacaat
ctggtacatg tccatctgat gttgttaata aggtagaggg caccacgtta 120
gcagataaaa tattgcaatg gtcaagcctt ggtatatttt tgggtggact tggcataggt
180 actggaagtg gtacaggggg tcgtacaggg tacattccat tgggtgggcg
ttccaataca 240 gttgtggatg tcggtcctac acgtcctcca gtggttattg
aacctgtggg ccccacagac 300 ccatctattg ttacattaat agaggactca
agtgttgtta catcaggtgc acctaggcct 360 acttttactg gcacgtctgg
gtttgatata acatctgctg gtacaactac acctgcagtt 420 ttggatatca
caccttcgtc tacctctgtt tctatttcca caaccaattt taccaatcct 480
gcattttctg atccgtccat tattgaagtt ccacaaactg gggaggtgtc aggtaatgta
540 tttgttggta cccctacatc tggaacacat gggtatgaag aaataccttt
acaaacattt 600 gcttcttctg gtacggggga ggaacccatt agtagtaccc
cattgcctac tgtgcggcgt 660 gtagcaggtc cccgccttta cagtagggcc
taccaacaag tgtctgtggc taaccctgag 720 tttcttacac gtccatcctc
tttaattacc tatgacaacc cggcctttga gcctgtggac 780 actacattaa
catttgagcc tcgtagtaat gttcctgatt cagattttat ggatattatc 840
cgtttacata ggcctgcttt aacatccagg cgtggtactg tgcgctttag tagattaggt
900 caaagggcaa ctatgtttac ccgtagcggt acacaaatag gtgctagggt
tcacttttat 960 catgatataa gtcctattgc accctcccca gaatatattg
aactgcagcc tttagtatct 1020 gccacggagg acaatggctt gtttgatata
tatgcagatg acatagaccc tgcaatgcct 1080 gtaccatcgc gtcctactac
ctcctctgca gtttctacat attcgcccac tatatcatct 1140 gcctcttcct
atagtaatgt aacggtccct ttaacctcct cttgggatgt gcctgtatac 1200
acgggtcctg atattacatt accacctact acctctgtat ggcccattgt atcacccaca
1260 gcccctgcct ctacacagta tattggtata catggtacac attattattt
gtggccatta 1320 tattatttta ttcctaaaaa gcgtaaacgt gttccctatt
tttttgcaga tggctttgtg 1380 gcggcctag 1389 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 461
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: HPV18 L2
Consensus Sequence <400> SEQUENCE: 4 Met Val Ser His Arg Ala
Ala Arg Arg Lys Arg Ala Ser Val Thr Asp 1 5 10 15 Leu Tyr Lys Thr
Cys Lys Gln Ser Gly Thr Cys Pro Ser Asp Val Val 20 25 30 Asn Lys
Val Glu Gly Thr Thr Leu Ala Asp Lys Ile Leu Gln Trp Ser 35 40 45
Ser Leu Gly Ile Phe Leu Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly 50
55 60 Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Gly Arg Ser Asn
Thr 65 70 75 80 Val Val Asp Val Gly Pro Thr Arg Pro Pro Val Val Ile
Glu Pro Val 85 90 95 Gly Pro Thr Asp Pro Ser Ile Val Thr Leu Ile
Glu Asp Ser Ser Val 100 105 110 Val Thr Ser Gly Ala Pro Arg Pro Thr
Phe Thr Gly Thr Ser Gly Phe 115 120 125 Asp Ile Thr Ser Ala Gly Thr
Thr Thr Pro Ala Val Leu Asp Ile Thr 130 135 140 Pro Ser Ser Thr Ser
Val Ser Ile Ser Thr Thr Asn Phe Thr Asn Pro 145 150 155 160 Ala Phe
Ser Asp Pro Ser Ile Ile Glu Val Pro Gln Thr Gly Glu Val 165 170 175
Ser Gly Asn Val Phe Val Gly Thr Pro Thr Ser Gly Thr His Gly Tyr 180
185 190 Glu Glu Ile Pro Leu Gln Thr Phe Ala Ser Ser Gly Thr Gly Glu
Glu 195 200 205 Pro Ile Ser Ser Thr Pro Leu Pro Thr Val Arg Arg Val
Ala Gly Pro 210 215 220 Arg Leu Tyr Ser Arg Ala Tyr Gln Gln Val Ser
Val Ala Asn Pro Glu 225 230 235 240 Phe Leu Thr Arg Pro Ser Ser Leu
Ile Thr Tyr Asp Asn Pro Ala Phe 245 250 255 Glu Pro Val Asp Thr Thr
Leu Thr Phe Glu Pro Arg Ser Asn Val Pro 260 265 270 Asp Ser Asp Phe
Met Asp Ile Ile Arg Leu His Arg Pro Ala Leu Thr 275 280 285 Ser Arg
Arg Gly Thr Val Arg Phe Ser Arg Leu Gly Gln Arg Ala Thr 290 295 300
Met Phe Thr Arg Ser Gly Thr Gln Ile Gly Ala Arg Val His Phe Tyr 305
310 315 320 His Asp Ile Ser Pro Ile Ala Pro Ser Pro Glu Tyr Ile Glu
Leu Gln 325 330 335 Pro Leu Val Ser Ala Thr Glu Asp Asn Gly Leu Phe
Asp Ile Tyr Ala 340 345 350 Asp Asp Ile Asp Pro Ala Met Pro Val Pro
Ser Arg Pro Thr Thr Ser 355 360 365 Ser Ala Val Ser Thr Tyr Ser Pro
Thr Ile Ser Ser Ala Ser Ser Tyr 370 375 380 Ser Asn Val Thr Val Pro
Leu Thr Ser Ser Trp Asp Val Pro Val Tyr 385 390 395 400 Thr Gly Pro
Asp Ile Thr Leu Pro Pro Thr Ser Val Trp Pro Ile Val 405 410 415 Ser
Pro Thr Ala Pro Ala Ser Thr Gln Tyr Ile Gly Ile His Gly Thr 420 425
430 His Tyr Tyr Leu Trp Pro Leu Tyr Tyr Phe Ile Pro Lys Lys Arg Lys
435 440 445 Arg Val Pro Tyr Phe Phe Ala Asp Gly Phe Val Ala Ala 450
455 460 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
5 <211> LENGTH: 41 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: oligonucleotide, sense primer <400>
SEQUENCE: 5 gaagatctca caaaacaaaa tggctttgtg gcggcctagt g 41
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: oligonucleotide, antisense primer <400>
SEQUENCE: 6 gaagatcttt acttcctggc acgtacacgc acacgc 36 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211>
LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, sense primer <400> SEQUENCE: 7 tcccccgggc
acaaaacaaa atggtatccc accgtgccgc acgac 45 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, antisense primer <400> SEQUENCE: 8
tcccccgggc taggccgcca caaagccatc tgc 33 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 9 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, sense primer <400> SEQUENCE: 9
caatccttat atattaaagg cacaggtatg 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, antisense primer <400> SEQUENCE: 10
catcatattg cccaggtaca ggagactgtg 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 11 <211> LENGTH: 41
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, PCR primer <400> SEQUENCE: 11 gaagatctca
caaaacaaaa tggctttgtg gcggcctagt g 41 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, PCR primer <400> SEQUENCE: 12 cctaacgtcc
tcagaaacat tagac 25 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide, sense primer
<400> SEQUENCE: 13 cttaaagctt atgtcacttt ctcttgtatc 30
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: oligonucleotide, antisense primer <400>
SEQUENCE: 14 tgataagctt gctcaatggt tctcttcctc 30 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
oligonucleotide, PCR primer <400> SEQUENCE: 15 tggtcatccc
aaatcttgaa a 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 16 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide, PCR primer
<400> SEQUENCE: 16 caccgtagtg tttggaagcg a 21
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